N-pyrazole A2A receptor agonists

ABSTRACT

This specification discloses 2-adenosine N-pyrazole compounds having the following formula: 
                         
and methods for using the compounds as A2A receptor agonists to stimulate mammalian coronary vasodilatation for therapeutic purposes and for purposes of imaging the heart.

This application is a continuation of U.S. application Ser. No.10/652,378, filed on Aug. 29, 2003, which is a continuation of U.S.application Ser. No. 10/018,446, filed on Apr. 12, 2002, now U.S. Pat.No. 6,642,210, which is a 371 ofPCT/US00/40281, filed on Jun. 21, 2000,which is a continuation-in-part of U.S. application Ser. No. 09/338,185,filed on Jun. 22, 1999, now U.S. Pat. No. 6,403,567, the specificationsof each of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention includes N-pyrazole substituted 2-adenosine compoundsthat are useful as A_(2A) receptor agonists. The compounds of thisinvention are vasodilating agents that are useful as heart imaging aidsthat aid in the identification of mammals, and especially humans who aresuffering from coronary disorders such poor coronary perfusion which isindicative of coronary artery disease (CAD). The compounds of thisinvention can also be used as therapeutics for coronary artery diseaseas well as any other disorders mediated by the A_(2A) receptor.

2. Description of the Art

Pharmacological stress is frequently induced with adenosine ordipyridamole in patients with suspected CAD before imaging with T1scintigraphy or echocardiography. Both drugs effect dilation of thecoronary resistance vessels by activation of cell surface A₂ receptors.Although pharmacological stress was originally introduced as a mean ofprovoking coronary dilation in patients unable to exercise, severalstudies have shown that the prognostic value of ²⁰¹T1 orechocardiographic imaging in patients subjected to pharmacologicalstress with adenosine or dipyridamole was equivalent to patientssubjected to traditional exercise stress tests. However, there is a highincidence of drug-related adverse side effects during pharmacologicalstress imaging with these drugs such as headache and nausea, that couldbe improved with new therapeutic agents.

Adenosine A_(2B) and A3 receptors are involved in a mast celldegranulation and, therefore, asthmatics are not give the non-specificadenosine agonists to induce a pharmacological stress test.Additionally, adenosine stimulation of the A₁ receptor in the atrium andA-V node will diminish the S—H interval which can induce AV block (N. C.Gupto et al.; J. Am Coll. Cardiol; (1992) 19: 248–257). Also,stimulation of the adenosine A₁ receptor by adenosine may be responsiblefor the nausea since the A₁ receptor is found in the intestinal tract(J. Nicholls et al.; Eur. J. Pharm.(1997) 338(2) 143–150).

Animal data suggests that specific adenosine A_(2A) subtype receptors oncoronary resistance vessels mediate the coronary dilatory responses toadenosine, whereas subtype A_(2B) receptor stimulation relaxesperipheral vessels (note: the latter lowers systemic blood pressure). Asa result there is a need for pharmaceutical compositions that are A_(2A)receptor agonists that have no pharmacological effect as a result ofstimulating the A₁ receptor in vivo. Furthermore, there is a need forA_(2A) receptor agonists that have a short half-life, and that are welltolerated by patients undergoing pharmacological coronary stressevaluations.

SUMMARY OF THE INVENTION

In one aspect, this invention includes 2-adenosine N-pyrazole compoundsthat are useful A_(2A) receptor agonists.

In another aspect, this invention includes pharmaceutical compoundsincluding 2-adenosine N-pyrazole that are well tolerated with few sideeffects.

Still another aspect of this invention are N-pyrazole compounds that canbe easily used in conjunction with radioactive imaging agents tofacilitate coronary imaging.

In one embodiment, this invention includes 2-adenosine N-pyrazolecompounds having the following formula:

In another embodiment, this invention includes methods for usingcompounds of this invention to stimulate coronary vasodilatation inmammals, and especially in humans, for stressing the heart induced stealsituation for purposes of imaging the heart.

In still another embodiment, this invention is a pharmaceuticalcomposition comprising one or more compounds of this invention and oneor more pharmaceutical excipients.

DESCRIPTION OF THE FIGURES

FIG. 1A is a analog record of the increase in coronary conductancecaused by Compound 16 of this invention before and after infusions ofCPX and ZM241385;

FIG. 1B is a summary of the data shown in FIG. 1A showing that CPX didnot but that ZM241385 did attenuate the increase in coronary conductancecaused by Compound 16 of this invention. In FIG. 1B, the bars representmean ±SEM of single measurement from 6 rat isolated perfused hearts;

FIG. 2 is a concentration response curve for the A₁ adenosine receptor(AdoR)-mediated negative dromotropic (AV conduction time) and A_(2A)AdoR-mediated vasodialator (increase coronary conductance) effects ofCompound 16 in rat isolated perfused hearts. Symbols and error barsindicate means ±SEM of single determination from each of four hearts.EC₅₀ value (potency) is the concentration of Compound 16 that causes 50%of maximal response;

FIG. 3 is a concentration response curve for the A₁ adenosine receptor(AdoR)-mediated negative dromotropic (AV conduction time) and A_(2A)AdoR-mediated vasodialator (increase coronary conductance) effects ofCompound 16 in guinea pig isolated perfused hearts. Symbols and errorbars indicate means ±SEM of single determination from each of fourhearts. EC₅₀ value (potency) is the concentration of Compound 16 thatcauses 50% of maximal response; and

FIG. 4 is a plot of the effect of CVT510, an A₁ adenosine receptoragonist and Compound 16 of this invention, an A_(2A) adenosine receptoragonist on atrioventricular (AV) conduction time in rat isolatedperfused hearts.

DESCRIPTION OF THE CURRENT EMBODIMENT

This invention includes a new class of 2-adenosine N-pyrazoles havingthe formula:

wherein R¹═CH₂OH, —CONR₅R₆;

R³ is independently selected from the group consisting of C₁₋₁₅ alkyl,halo, NO₂, CF₃, CN, OR²⁰, SR²⁰, N(R²⁰)₂, S(O)R²², SO₂R²², SO₂N(R²⁰)₂,SO₂NR²⁰COR²², SO₂NR²⁰CO₂R²², SO₂NR²⁰CON(R²⁰)₂, N(R²⁰)₂ NR²⁰COR²²,NR²⁰CO₂R²², NR²⁰CON(R²⁰)₂, NR²⁰C(NR²⁰)NHR²³, COR²⁰, CO₂R²⁰, CON(R²⁰)₂,CONR²⁰SO₂R²², NR²⁰SO₂R²², SO₂NR²⁰CO₂R²², OCONR²⁰SO₂R²², OC(O)R²⁰,C(O)OCH₂OC(O)R²⁰, and OCON(R²⁰)₂, —CONR⁷R⁸, C₂₋₁₅ alkenyl, C₂₋₁₅alkynyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl, alkenyl,alkynyl, aryl, heterocyclyl and heteroaryl substituents are optionallysubstituted with from 1 to 3 substituents independently selected fromthe group consisting of halo, alkyl, NO₂, heterocyclyl, aryl,heteroaryl, CF₃, CN, OR²⁰, SR²⁰, N(R²⁰)₂, S(O)R²², SO₂R²², SO₂N(R²⁰)₂,SO₂NR²⁰COR²², SO₂NR²⁰CO₂R²², SO₂NR²⁰CON(R²⁰)₂, N(R²⁰)₂ NR²⁰COR²²,NR²⁰CO₂R²², NR²⁰CON(R²⁰)₂, COR²⁰, CO₂R²⁰, CON(R²⁰)₂, CONR²⁰SO₂R²²,NR²⁰SO₂R²², SO₂NR²⁰CO₂R²², OCONR²⁰SO₂R²², OC(O)R²⁰, C(O)OCH₂OC(O)R²⁰,and OCON(R²⁰)₂ and wherein the optionally substituted heteroaryl, aryl,and heterocyclyl substituents are optionally substituted with halo, NO₂,alkyl, CF₃, amino, mono- or di-alkylamino, alkyl or aryl or heteroarylamide, NCOR²², NR²⁰SO₂R²², COR²⁰, CO₂R²⁰, CON(R²⁰)₂, NR²⁰CON(R²⁰)₂,OC(O)R²⁰, OC(O)N(R²⁰)₂, SR²⁰, S(O)R²², SO₂R²², SO₂N(R²⁰)₂, CN, or OR²⁰;

R⁵ and R⁶ are each individually selected from H, and C₁–C₁₅ alkyl thatis optionally substituted with from 1 to 2 substituents independentlyselected from the group of halo, NO₂, heterocyclyl, aryl, heteroaryl,CF₃, CN, OR²⁰, SR²⁰, N(R²⁰)₂, S(O)R²², SO₂R²², SO₂N(R²⁰)₂, SO₂NR²⁰COR²²,SO₂NR²⁰CO₂R²², SO₂NR²⁰CON(R²⁰)₂, N(R²⁰)₂ NR²⁰COR²², NR²⁰CO₂R²²,NR²⁰CON(R²⁰)₂, COR²⁰, CO₂R²⁰, CON(R²⁰)₂, CONR²⁰SO₂R²², NR²⁰SO₂R²²,SO₂NR²⁰CO₂R²², OCONR²⁰SO₂R²², OC(O)R²⁰, C(O)OCH₂OC(O)R²⁰, and OCON(R²⁰)₂wherein each optionally substituted heteroaryl, aryl, and heterocyclylsubstituent is optionally substituted with halo, NO₂, alkyl, CF₃, amino,monoalkylamino, dialkylamino, alkylamide, arylamide, heteroarylamide,NCOR²², NR²⁰SO₂R²², COR²⁰, CO₂R²⁰, CON(R²⁰)₂, NR²⁰CON(R²⁰)₂, OC(O)R²⁰,OC(O)N(R²⁰)₂, SR²⁰, S(O)R²², SO₂R²², SO₂N(R²⁰)₂, CN, and OR²⁰;

R⁷ is selected from the group consisting of hydrogen, C₁₋₁₅ alkyl, C₂₋₁₅alkenyl, C₂₋₁₅ alkynyl, heterocyclyl, aryl and heteroaryl, wherein thealkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl substituentsare optionally substituted with from 1 to 3 substituents independentlyselected from the group of halo, NO₂, heterocyclyl, aryl, heteroaryl,CF₃, CN, OR²⁰, SR²⁰, N(R²⁰)₂, S(O)R²², SO₂R²², SO₂N(R²⁰)₂, SO₂NR²⁰COR²²,SO₂NR²⁰CO₂R²², SO₂NR²⁰CON(R²⁰)₂, N(R²⁰)₂ NR²⁰COR²², NR²⁰CO₂R²²,NR²⁰CON(R²⁰)₂, COR²⁰, CO₂R²⁰, CON(R²⁰)₂, CONR²⁰SO₂R²², NR²⁰SO₂R²²,SO₂NR²⁰CO₂R²², OCONR²⁰SO₂R²², OC(O)R²⁰, C(O)OCH₂OC(O)R²⁰ and OCON(R²⁰)₂and wherein each optionally substituted heteroaryl, aryl andheterocyclyl substituent is optionally substituted with halo, NO₂,alkyl, CF₃, amino, mono- or di-alkylamino, alkyl or aryl or heteroarylamide, NCOR²², NR²²SO₂R²², COR²⁰, CO₂R²⁰, CON(R²⁰)₂, NR²⁰CON(R²⁰)₂,OC(O)R²⁰, OC(O)N(R²⁰)₂, SR²⁰, S(O)R²², SO₂R²², SO₂N(R²⁰)₂, CN, and OR²⁰;

R⁸ is selected from the group consisting of hydrogen, C₁₋₁₅ alkyl, C₂₋₁₅alkenyl, C₂₋₁₅ alkynyl, heterocyclyl, aryl, and heteroaryl, wherein thealkyl, alkenyl, alkynyl, aryl, heterocyclyl, and heteroaryl substituentsare optionally substituted with from 1 to 3 substituents independentlyselected from the group consisting of halo, NO₂, heterocyclyl, aryl,heteroaryl, CF₃, CN, OR²⁰, SR²⁰, N(R²⁰)₂, S(O)R²², SO₂R²², SO₂N(R²⁰)₂,SO₂NR²⁰COR²², SO₂NR²⁰CO₂R²², SO₂NR²⁰CON(R²⁰)₂, N(R²⁰)₂ NR²⁰COR²²,NR²⁰CO₂R²², NR²⁰CON(R²⁰)₂, COR²⁰, CO₂R²⁰, CON(R²⁰)₂, CONR²⁰SO₂R²²,NR²⁰SO₂R²², SO₂NR²⁰CO₂R²², OCONR²⁰SO₂R²², OC(O)R²⁰, C(O)OCH₂OC(O)R²⁰,and OCON(R²⁰)₂ and wherein each optionally substituted heteroaryl, aryl,and heterocyclyl substituent is optionally substituted with halo, NO₂,alkyl, CF₃, amino, mono- or di-alkylamino, alkyl or aryl or heteroarylamide, NCOR²², NR²SO₂R²², COR²⁰, CO₂R²⁰, CON(R²⁰)₂, NR²⁰CON(R²⁰)₂,OC(O)R²⁰, OC(O)N(R²⁰)₂, SR²⁰, S(O)R²², SO₂R²², SO₂N(R²⁰)₂, CN, and OR²⁰;

R²⁰ is selected from the group consisting of H, C₁₋₁₅ alkyl, C₂₋₁₅alkenyl, C₂₋₁₅ alkynyl, heterocyclyl, aryl, and heteroaryl, wherein thealkyl, alkenyl, alkynyl, heterocyclyl, aryl, and heteroaryl substituentsare optionally substituted with from I to 3 substituents independentlyselected from halo, alkyl, mono- or dialkylamino, alkyl or aryl orheteroaryl amide, CN, O—C₁₋₆ alkyl, CF₃, aryl, and heteroaryl;

R²² is selected from the group consisting of C₁₋₁₅ alkyl, C₂₋₁₅ alkenyl,C₂₋₁₅ alkynyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl,alkenyl, alkynyl, heterocyclyl, aryl, and heteroaryl substituents areoptionally substituted with from 1 to 3 substituents independentlyselected from halo, alkyl, mono- or dialkylamino, alkyl or aryl orheteroaryl amide, CN, O—C₁₋₆ alkyl, CF₃, aryl, and heteroaryl; and

wherein R² and R⁴ are selected from the group consisting of H, C₁₋₆alkyl and aryl, wherein the alkyl and aryl substituents are optionallysubstituted with halo, CN, CF₃, OR²⁰ and N(R²⁰)₂ with the proviso thatwhen R² is not hydrogen then R⁴ is hydrogen, and when R⁴ is not hydrogenthen R² is hydrogen.

In preferred compounds of this invention, R³ is selected from the groupconsisting of C₁₋₁₅ alkyl, halo,CF₃, CN, OR²⁰, SR²⁰, S(O)R²², SO₂R²²,SO₂N(R²⁰)₂, COR²⁰, CO₂R²⁰, —CONR⁷R⁸, aryl and heteroaryl wherein thealkyl, aryl and heteroaryl substituents are optionally substituted withfrom 1 to 3 substituents independently selected from the groupconsisting of halo, aryl, heteroaryl, CF₃, CN, OR²⁰, SR²⁰, S(O)R²²,SO₂R²², SO₂N(R²⁰)₂, COR²⁰, CO₂R²⁰ or CON(R²⁰)₂, and each optionalheteroaryl and aryl substituent is optionally substituted with halo,alkyl, CF₃ CN, and OR²⁰; R⁵ and R⁶ are independently selected from thegroup of H and C₁–C₁₅ alkyl including one optional aryl substituent andeach optional aryl substituent is optionally substituted with halo orCF₃; R⁷ is selected from the group consisting of C₁₋₁₅ alkyl, C₂₋₁₅alkynyl, aryl, and heteroaryl, wherein the alkyl, alkynyl, aryl, andheteroaryl substituents are optionally substituted with from 1 to 3substituents independently selected from the group consisting of halo,aryl, heteroaryl, CF₃, CN, OR²⁰, and each optional heteroaryl and arylsubstituent is optionally substituted with halo, alkyl, CF₃ CN, or OR²⁰;R⁸ is selected from the group consisting of hydrogen and C₁₋₁₅ alkyl;R²⁰ is selected from the group consisting of H, C₁₋₄ alkyl and aryl,wherein alkyl and aryl substituents are optionally substituted with onealkyl substituent; and R²² is selected from the group consisting of C₁₋₄alkyl and aryl which are each optionally substituted with from 1 to 3alkyl group.

In more preferred compounds, R¹ is CH₂OH; R³ is selected from the groupconsisting of CO₂R²⁰, —CONR⁷R⁸ and aryl where the aryl substituent isoptionally substituted with from 1 to 2 substituents independentlyselected from the group consisting of halo, C₁₋₆ alkyl, CF₃ and OR²⁰; R⁷is selected from the group consisting of hydrogen, C₁₋₈ alkyl and aryl,where the alkyl and aryl substituents are optionally substituted withone substituent selected from the group consisting of halo, aryl, CF₃,CN, OR²⁰ and wherein each optional aryl substituent is optionallysubstituted with halo, alkyl, CF₃ CN, and OR²⁰; R⁸ is selected from thegroup consisting of hydrogen and C₁₋₈ alkyl; and R²⁰ is selected fromhydrogen and C₁₋₄ alkyl.

In a still more preferred embodiment, R¹═CH₂OH; R³ is selected from thegroup consisting of CO₂R²⁰, —CONR⁷R⁸, and aryl that is optionallysubstituted with one substituent selected from the group consisting ofhalo, C₁₋₃ alkyl and OR²⁰; R⁷ is selected from hydrogen, and C₁₋₃ alkyl;R⁸ is hydrogen; and R²⁰ is selected from hydrogen and C₁₋₄ alkyl. Inthis preferred embodiment, R³ is most preferably selected from —CO₂Etand —CONHEt.

In another still more preferred embodiment, R¹═—CONHEt, R³ is selectedfrom the group consisting of CO₂R²⁰, —CONR⁷R⁸, and aryl wherein the arylsubstituent is optionally substituted with from 1 to 2 substituentsindependently selected from the group consisting of halo, C₁₋₃ alkyl,CF₃ or OR²⁰; R⁷ is selected from the group consisting of hydrogen, andC₁₋₈ alkyl that is optionally substituted with one substituent selectedfrom the group consisting of halo, CF₃, CN or OR²⁰; R⁸ is selected fromthe group consisting of hydrogen and C₁₋₃ alkyl; and R²⁰ is selectedfrom the group consisting of hydrogen and C₁₋₄ alkyl. In this morepreferred embodiment, R⁸ is preferably hydrogen, R⁷ is preferablyselected from the group consisting of hydrogen, and C₁₋₃ _(—) alkyl, andR²⁰ is preferably selected from the group consisting of hydrogen andC₁₋₄ alkyl.

In a most preferred embodiment, the compound of this invention isselected fromethyl1{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate,(4S,2R,3R,5R)-2-{6-amino-2-[4-(4-chlorophenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol,(4S,2R,3R,5R)-2-{6-amino-2-[4-(4-methoxyphenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol,(4S,2R,3R,5R)-2-{6-amino-2-[4-(4-methylphenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol,(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide,1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylicacid,(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N,N-dimethylcarboxamide,(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-ethylcarboxamide,1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxamide,1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-(cyclopentylmethyl)carboxamide,(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-[(4-chlorophenyl)methyl]carboxamide,Ethyl 2-[(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)carbonylamino]acetate,and mixtures thereof.

The following definitions apply to terms as used herein.

“Halo” or “Halogen”—alone or in combination means all halogens, that is,chloro (Cl), fluoro (F), bromo (Br), iodo (I).

“Hydroxyl” refers to the group —OH.

“Thiol” or “mercapto” refers to the group —SH.

“Alkyl”—alone or in combination means an alkane-derived radicalcontaining from 1 to 20, preferably 1 to 15, carbon atoms (unlessspecifically defined). It is a straight chain alkyl, branched alkyl orcycloalkyl. Preferably, straight or branched alkyl groups containingfrom 1–15, more preferably 1 to 8, even more preferably 1–6, yet morepreferably 1–4 and most preferably 1–2, carbon atoms, such as methyl,ethyl, propyl, isopropyl, butyl, t-butyl and the like. The term “loweralkyl” is used herein to describe the straight chain alkyl groupsdescribed immediately above. Preferably, cycloalkyl groups aremonocyclic, bicyclic or tricyclic ring systems of 3–8, more preferably3–6, ring members per ring, such as cyclopropyl, cyclopentyl,cyclohexyl, adamantyl and the like. Alkyl also includes a straight chainor branched alkyl group that contains or is interrupted by a cycloalkylportion. The straight chain or branched alkyl group is attached at anyavailable point to produce a stable compound. Examples of this include,but are not limited to, 4-(isopropyl)-cyclohexylethyl or2-methyl-cyclopropylpentyl. A substituted alkyl is a straight chainalkyl, branched alkyl, or cycloalkyl group defined previously,independently substituted with 1 to 3 groups or substituents of halo,hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy,aryloxy, heteroaryloxy, amino optionally mono- or di-substituted withalkyl, aryl or heteroaryl groups, amidino, urea optionally substitutedwith alkyl, aryl, heteroaryl or heterocyclyl groups, aminosulfonyloptionally N-mono- or N,N-di-substituted with alkyl, aryl or heteroarylgroups, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino,alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or thelike.

“Alkenyl”—alone or in combination means a straight, branched, or cyclichydrocarbon containing 2–20, preferably 2–17, more preferably 2–10, evenmore preferably 2–8, most preferably 2–4, carbon atoms and at least one,preferably 1–3, more preferably 1–2, most preferably one, carbon tocarbon double bond. In the case of a cycloalkyl group, conjugation ofmore than one carbon to carbon double bond is not such as to conferaromaticity to the ring. Carbon to carbon double bonds may be eithercontained within a cycloalkyl portion, with the exception ofcyclopropyl, or within a straight chain or branched portion. Examples ofalkenyl groups include ethenyl, propenyl, isopropenyl, butenyl,cyclohexenyl, cyclohexenylalkyl and the like. A substituted alkenyl isthe straight chain alkenyl, branched alkenyl or cycloalkenyl groupdefined previously, independently substituted with 1 to 3 groups orsubstituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono-or di-substituted with alkyl, aryl or heteroaryl groups, amidino, ureaoptionally substituted with alkyl, aryl, heteroaryl or heterocyclylgroups, aminosulfonyl optionally N-mono- or N,N-di-substituted withalkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino,heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, carboxy, alkoxycarbonyl, aryloxycarbonyl,heteroaryloxycarbonyl, or the like attached at any available point toproduce a stable compound.

“Alkynyl”—alone or in combination means a straight or branchedhydrocarbon containing 2–20, preferably 2–17, more preferably 2–10, evenmore preferably 2–8, most preferably 2–4, carbon atoms containing atleast one, preferably one, carbon to carbon triple bond. Examples ofalkynyl groups include ethynyl, propynyl, butynyl and the like. Asubstituted alkynyl refers to the straight chain alkynyl or branchedalkenyl defined previously, independently substituted with 1 to 3 groupsor substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono-or di-substituted with alkyl, aryl or heteroaryl groups, amidino, ureaoptionally substituted with alkyl, aryl, heteroaryl or heterocyclylgroups, aminosulfonyl optionally N-mono- or N,N-di-substituted withalkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino,heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, or the like attached at any available point toproduce a stable compound.

“Alkyl alkenyl” refers to a group —R—CR′═CR′″ R″″, where R is loweralkyl, or substituted lower alkyl, R′, R′″, R″″ may independently behydrogen, halogen, lower alkyl, substituted lower alkyl, acyl, aryl,substituted aryl, hetaryl, or substituted hetaryl as defined below.

“Alkyl alkynyl” refers to a groups —RC≡CR′ where R is lower alkyl orsubstituted lower alkyl, R′ is hydrogen, lower alkyl, substituted loweralkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl asdefined below.

“Alkoxy” denotes the group —OR, where R is lower alkyl, substitutedlower alkyl, acyl, aryl, substituted aryl, aralkyl, substituted aralkyl,heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, or substituted cycloheteroalkyl as defined.

“Alkylthio” denotes the group —SR, —S(O)_(n=1-2)—R, where R is loweralkyl, substituted lower alkyl, aryl, substituted aryl, aralkyl orsubstituted aralkyl as defined herein.

“Acyl” denotes groups —C(O)R, where R is hydrogen, lower alkylsubstituted lower alkyl, aryl, substituted aryl and the like as definedherein.

“Aryloxy” denotes groups —OAr, where Ar is an aryl, substituted aryl,heteroaryl, or substituted heteroaryl group as defined herein.

“Amino” denotes the group NRR′, where R and R′ may independently byhydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl,hetaryl, or substituted hetaryl as defined herein or acyl.

“Amido” denotes the group —C(O)NRR′, where R and R′ may independently byhydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl,hetaryl, substituted hetaryl as defined herein.

“Carboxyl” denotes the group —C(O)OR, where R is hydrogen, lower alkyl,substituted lower alkyl, aryl, substituted aryl, hetaryl, andsubstituted hetaryl as defined herein.

“Aryl”—alone or in combination means phenyl or naphthyl optionallycarbocyclic fused with a cycloalkyl of preferably 5–7, more preferably5–6, ring members and/or optionally substituted with 1 to 3 groups orsubstituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono-or di-substituted with alkyl, aryl or heteroaryl groups, amidino, ureaoptionally substituted with alkyl, aryl, heteroaryl or heterocyclylgroups, aminosulfonyl optionally N-mono- or N,N-di-substituted withalkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino,heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino,heteroarylcarbonylamino, or the like.

“Substituted aryl” refers to aryl optionally substituted with one ormore functional groups, e.g., halogen, lower alkyl, lower alkoxy,alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy,heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfamido and the like.

“Heterocycle” refers to a saturated, unsaturated, or aromaticcarbocyclic group having a single ring (e.g., morpholino, pyridyl orfuryl) or multiple condensed rings (e.g., naphthpyridyl, quinoxalyl,quinolinyl, indolizinyl or benzo[b]thienyl) and having at least onehetero atom, such as N, O or S, within the ring, which can optionally beunsubstituted or substituted with, e.g., halogen, lower alkyl, loweralkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl,aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfamido and the like.

“Heteroaryl”—alone or in combination means a monocyclic aromatic ringstructure containing 5 or 6 ring atoms, or a bicyclic aromatic grouphaving 8 to 10 atoms, containing one or more, preferably 1–4, morepreferably 1–3, even more preferably 1–2, heteroatoms independentlyselected from the group O, S, and N, and optionally substituted with 1to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, aminooptionally mono- or di-substituted with alkyl, aryl or heteroarylgroups, amidino, urea optionally substituted with alkyl, aryl,heteroaryl or heterocyclyl groups, aminosulfonyl optionally N-mono- orN,N-di-substituted with alkyl, aryl or heteroaryl groups,alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino,alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or thelike. Heteroaryl is also intended to include oxidized S or N, such assulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon ornitrogen atom is the point of attachment of the heteroaryl ringstructure such that a stable aromatic ring is retained. Examples ofheteroaryl groups are pyridinyl, pyridazinyl, pyrazinyl, quinazolinyl,purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, oxazolyl,thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl,tetrazolyl, imidazolyl, triazinyl, furanyl, benzofuryl, indolyl and thelike. A substituted heteroaryl contains a substituent attached at anavailable carbon or nitrogen to produce a stable compound.

“Heterocyclyl”—alone or in combination means a non-aromatic cycloalkylgroup having from 5 to 10 atoms in which from 1 to 3 carbon atoms in thering are replaced by heteroatoms of O, S or N, and are optionally benzofused or fused heteroaryl of 5–6 ring members and/or are optionallysubstituted as in the case of cycloalkyl. Heterocycyl is also intendedto include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of atertiary ring nitrogen. The point of attachment is at a carbon ornitrogen atom. Examples of heterocyclyl groups are tetrahydrofuranyl,dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl,dihydrobenzofuryl, dihydroindolyl, and the like. A substitutedhetercyclyl contains a substituent nitrogen attached at an availablecarbon or nitrogen to produce a stable compound.

“Substituted heteroaryl” refers to a heterocycle optionally mono or polysubstituted with one or more functional groups, e.g., halogen, loweralkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl,hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl,substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

“Aralkyl” refers to the group —R—Ar where Ar is an aryl group and R islower alkyl or substituted lower alkyl group. Aryl groups can optionallybe unsubstituted or substituted with, e.g., halogen, lower alkyl,alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl,aryloxy, heterocycle, substituted heterocycle, hetaryl, substitutedhetaryl, nitro, cyano, thiol, sulfamido and the like.

“Heteroalkyl” refers to the group —R-Het where Het is a heterocyclegroup and R is a lower alkyl group. Heteroalkyl groups can optionally beunsubstituted or substituted with e.g., halogen, lower alkyl, loweralkoxy, alkylthio, acetylene, amino, amido, carboxyl, aryl, aryloxy,heterocycle, substituted heterocycle, hetaryl, substituted hetaryl,nitro, cyano, thiol, sulfamido and the like.

“Heteroarylalkyl” refers to the group —R-HetAr where HetAr is anheteroaryl group and R lower alkyl or substituted lower alkyl.Heteroarylalkyl groups can optionally be unsubstituted or substitutedwith, e.g., halogen, lower alkyl, substituted lower alkyl, alkoxy,alkylthio, acetylene, aryl, aryloxy, heterocycle, substitutedheterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfamido and the like.

“Cycloalkyl” refers to a divalent cyclic or polycyclic alkyl groupcontaining 3 to 15 carbon atoms.

“Substituted cycloalkyl” refers to a cycloalkyl group comprising one ormore substituents with, e.g., halogen, lower alkyl, substituted loweralkyl, alkoxy, alkylthio, acetylene, aryl, aryloxy, heterocycle,substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano,thiol, sulfamido and the like.

“Cycloheteroalkyl” refers to a cycloalkyl group wherein one or more ofthe ring carbon atoms is replaced with a heteroatom (e.g., N, O, S orP).

Substituted cycloheteroalkyl” refers to a cycloheteroalkyl group asherein defined which contains one or more substituents, such as halogen,lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl,hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl,substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

“Alkyl cycloalkyl” denotes the group -R-cycloalkyl where cycloalkyl is acycloalkyl group and R is a lower alkyl or substituted lower alkyl.Cycloalkyl groups can optionally be unsubstituted or substituted withe.g. halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino,amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substitutedheterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol,sulfamido and the like.

“Alkyl cycloheteroalkyl” denotes the group -R-cycloheteroalkyl where Ris a lower alkyl or substituted lower alkyl. Cycloheteroalkyl groups canoptionally be unsubstituted or substituted with e.g. halogen, loweralkyl, lower alkoxy, alkylthio, amino, amido, carboxyl, acetylene,hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl,substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

The compounds of this invention can be prepared as outlined in Schemes1–4. Compounds having the general formula IV can be prepared as shown inScheme 1.

Compound I can be prepared by reacting compound 1 with appropriatelysubstituted 1,3-dicarbonyl in a mixture of ACOH and MEOH at 80° C.(Holzer et al., J. Heterocycl. Chem. (1993) 30, 865). Compound II, whichcan be obtained by reacting compound I with 2,2-dimethoxypropane in thepresence of an acid, can be oxidized to the carboxylic acid III, basedon structurally similar compounds using potassium permanganate orpyridinium chlorochromate (M. Hudlicky, (1990) Oxidations in OrganicChemistry, ACS Monographs, American Chemical Society, Washington D. C.).Reaction of a primary or secondary amine having the formula HNR⁶R⁷, andcompound III using DCC (M. Fujino et al., Chem. Pharm. Bull. (1974), 22,1857), PYBOP (J. Martinez et al., J. Med. Chem. (1988) 28, 1874) orPyBrop (J. Caste et al. Tetrahedron, (1991), 32, 1967) couplingconditions can afford compound IV.

Compound V can be prepared as shown in Scheme 2. The Tri TBDMSderivative 4 can be obtained by treating compound 2 with TBDMSCI andimidazole in DMF followed by hydrolysis of the ethyl ester using NaOH.Reaction of a primary or secondary amine with the formula HNR⁶R⁷, andcompound 4 using DCC (M. Fujino et al., Chem. Pharm. Bull. (1974), 22,1857), PyBOP (J. Martinez et al., J. Med. Chem. (1988) 28, 1874) orPyBrop (J. Caste et al. Tetrahedron, (1991), 32, 1967) couplingconditions can afford compound V.

A specific synthesis of Compound 11 is illustrated in Scheme 3.Commercially available guanosine 5 was converted to the triacetate 6 aspreviously described (M. J. Robins and B. Uznanski, Can. J. Chem.(1981), 59, 2601–2607). Compound 7, prepared by following the literatureprocedure of Cerster et al. (J. F. Cerster, A. F. Lewis, and R. K.Robins, Org. Synthesis, 242–243), was converted to compound 9 in twosteps as previously described (V. Nair et al., J. Org. Chem., (1988),53, 3051–3057). Compound 1 was obtained by reacting hydrazine hydratewith compound 9 in ethanol at 80° C. Condensation of compound 1 withethoxycarbonylmalondialdehyde in a mixture of AcOH and MeOH at 80° C.produced compound 10. Heating compound 10 in excess methylamine affordedcompound 11.

The synthesis of 1,3-dialdehyde VII is described in Scheme 4. Reactionof 3,3-diethoxypropionate or 3,3-diethoxypropionitrile or1,1-diethoxy-2-nitroethane VI (R₃═CO₂R, CN or NO₂) with ethyl or methylformate in the presence of NaH can afford the dialdehyde VII (Y.Yamamoto et al., J. Org. Chem. (1989) 54, 4734).

Compounds of this invention are useful in conjunction with radioactiveimaging agents to image coronary activity. The compounds of thisinvention are A_(2A) agonists that are believed to provide specificactivation of adenosine A_(2A) receptors in the coronary vessels asopposed to adenosine A1 receptors in the atrium and AV-node and/orA_(2B) receptors in peripheral vessels, thus avoiding undesirableside-effects. Upon administration in a therapeutic amount, the compoundsof this invention cause coronary blood vessels to vasodilate to inducecoronary steal wherein healthy coronary vessels steal blood fromunhealthy vessels resulting in lack of blood flow to heart tissues.Lower doses of the A_(2A) agonists may provide beneficial coronaryvasodilatation (less severe) in the treatment of chronic CAD.

As A_(2A) agonists, the compounds of this invention are also useful inadjunctive therapy with angioplasty to induce dilation, inhibit plateletaggregation, and as a general anti-inflammatory agent. A_(2A) agonists,such as the compounds of this invention, can provide the therapeuticbenefits described above by preventing neutrophil activation (PurinergicApproaches in Experimental Therapeutics K. A. Jacobson and M. F. Jarvis1997 Wiley, N.Y.). The compounds of this invention are also effectiveagainst a condition called no-reflow in which platelets and neutrophilsaggregate and block a vessel. As A_(2A) agonists, the compounds of thisinvention are effective against no-reflow by preventing neutrophil andplatelet activation (e.g., they are believed to prevent release ofsuperoxide from neutrophils). As A_(2A) agonists, the compounds of thisinvention are also useful as cardioprotective agents through theiranti-inflammatory action on neutrophils. Thus, in situations when theheart will go through an ischemic state such as a transplant, they willbe useful.

This invention also includes pro-drugs of the above-identified A_(2A)agonists. A pro-drug is a drug which has been chemically modified andmay be biological inactive at its site of action, but which will bedegraded or modified by one or more enzymatic or in vivo processes tothe bioactive form. The pro-drugs of this invention should have adifferent pharmacokinetic profile to the parent enabling improvedabsorption across the mucosal epithelium, better salt formulation and/orsolubility and improved systemic stability. The above-identifiedcompounds may be preferably modified at one or more of the hydroxylgroups. The modifications may be (1) ester or carbamate derivativeswhich may be cleaved by esterases or lipases, for example; (2) peptideswhich may be recognized by specific or non specific proteinase; or (3)derivatives that accumulate at a site of action through membraneselection or a pro-drug form or modified pro-drug form, or anycombination of (1) to (3) above.

The compounds may be administered orally, intravenously, through theepidermis or by any other means known in the art for administering atherapeutic agents. The method of treatment comprises the administrationof an effective quantity of the chosen compound, preferably dispersed ina pharmaceutical carrier. Dosage units of the active ingredient aregenerally selected from the range of 0.01 to 100 mg/kg, but will bereadily determined by one skilled in the art depending upon the route ofadministration, age and condition of the patient. This dose is typicallyadministered in a solution about 5 minutes to about an hour or moreprior to coronary imaging. No unacceptable toxicological effects areexpected when compounds of the invention are administered in accordancewith the present invention.

If the final compound of this invention contains a basic group, an acidaddition salt may be prepared. Acid addition salts of the compounds areprepared in a standard manner in a suitable solvent from the parentcompound and an excess of acid, such as hydrochloric, hydrobromic,sulfuric, phosphoric, acetic, maleic, succinic, or methanesulfonic. Thehydrochloric salt form is especially useful. If the final compoundcontains an acidic group, cationic salts may be prepared. Typically theparent compound is treated with an excess of an alkaline reagent, suchas hydroxide, carbonate or alkoxide, containing the appropriate cation.Cations such as Na⁺, K⁺, Ca⁺² and NH₄ ⁺ are examples of cations presentin pharmaceutically acceptable salts. Certain of the compounds forminner salts or zwitterions which may also be acceptable.

Pharmaceutical compositions including the compounds of this invention,and/or derivatives thereof, may be formulated as solutions orlyophilized powders for parenteral administration. Powders may bereconstituted by addition of a suitable diluent or otherpharmaceutically acceptable carrier prior to use. If used in liquid formthe compositions of this invention are preferably incorporated into abuffered, isotonic, aqueous solution. Examples of suitable diluents arenormal isotonic saline solution, standard 5% dextrose in water andbuffered sodium or ammonium acetate solution. Such liquid formulationsare suitable for parenteral administration, but may also be used fororal administration. It may be desirable to add excipients such aspolyvinylpyrrolidinone, gelatin, hydroxycellulose, acacia, polyethyleneglycol, mannitol, sodium chloride, sodium citrate or any other excipientknown to one of skill in the art to pharmaceutical compositionsincluding compounds of this invention. Alternatively, the pharmaceuticalcompounds may be encapsulated, tableted or prepared in an emulsion orsyrup for oral administration. Pharmaceutically acceptable solid orliquid carriers may be added to enhance or stabilize the composition, orto facilitate preparation of the composition. Liquid carriers includesyrup, peanut oil, olive oil, glycerin, saline, alcohols and water.Solid carriers include starch, lactose, calcium sulfate, dihydrate,teffa alba, magnesium stearate or stearic acid, talc, pectin, acacia,agar or gelatin. The carrier may also include a sustained releasematerial such as glycerol monostearate or glycerol distearate, alone orwith a wax. The amount of solid carrier varies but, preferably, will bebetween about 20 mg to about 1 gram per dosage unit. The pharmaceuticaldosages are made using conventional techniques such as milling, mixing,granulation, and compressing, when necessary, for tablet forms; ormilling, mixing and filling for hard gelatin capsule forms. When aliquid carrier is used, the preparation will be in the form of a syrup,elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquidformulation may be administered directly or filled into a soft gelatincapsule. It is preferred that the compositions of this invention areadministered as a solution either orally or intravenously by continuosinfusion or bolus.

The Examples which follow serve to illustrate this invention. TheExamples are intended to in no way limit the scope of this invention,but are provided to show how to make and use the compounds of thisinvention. In the Examples, all temperatures are in degrees Centigrade.

EXAMPLE 1

ethyl1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylate(12)

To a suspension of 2-hydrazinoadenosine (0.025 g, 0.08 mmol) in a 1:1mixture of MeOH/AcOH was added (ethoxycarbonyl)malondialdehyde ((0.019g, 0.12 mmol) and the mixture was heated at 80° C. for 3 h. Theprecipitate formed was collected by filtration and washed with EtOH andether to afford 12. ¹HNMR (DMSO-d6) δ1.25 (t, 3H), 3.5 (m, 1H), 3.6 (m,1H), 3.8 (d, 1H), 4.15 (d, 1H), 4.55 (m, 1H), 5.0 (t, 1H), 5.2 (d, 1H),5.5 (d, 1H), 5.9 (d, 1H), 7.15–7.3 (m, 5H), 7.8 (br s, 2H), 8.1 (s, 1H),8.4 (s, 1H), 8.9 (s, 1H).

EXAMPLE 2

(4S,2R,3R,5R)-2-{6-amino-2-[4-(4-chlorophenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol(13)

To a suspension of 2-hydrazinoadenosine (0.025 g, 0.08 mmol) in a 1:1mixture of MeOH/AcOH was added 2-(4-chloro)phenylmalondialdehyde(0.022g, 0.12 mmol) and the mixture was heated at 80° C. for 3 h. Theprecipitate formed was collected by filtration and washed with EtOH andEther to afford 13. ¹HNMR (DMSO-d6) 63.5 (m, 1H), 3.6 (m, 1H), 3.8 (d,1H), 4.15 (d, 1H), 4.2 (q, 2H), 4.55 (m, 1H), 5.9 (d, 1H), 7.45 (d 2H),7.75 (d, 2H), 8.25 (s, 1H), 8.35 (s, 1H), 8.9 (s, 1H).

EXAMPLE 3

(4S,2R,3R,5R)-2-{6-amino-2-[4-(4-methoxyphenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol(14)

To a suspension of 2-hydrazinoadenosine (0.025 g, 0.08 mmol) in a 1:1mixture of MeOH/AcOH was added 2-(4-methoxy)phenylmalondialdehyde(0.022g, 0.12 mmol) and the mixture was heated at 80° C. for 3 h. Theprecipitate formed was collected by filtration and washed with EtOH andEther to afford 14. ¹HNMR (DMSO-d6) δ3.55 (m, 1H), 3.65 (m, 1H), 3.75(s, 3H), 3.9 (d, 1H), 4.15 (d, 1H), 4.6 (m, 1H), 5.9 (d, 1H), 6.75 (d,2H), 7.6 (d, 2H), 8.15 (s, 1H), 8.35 (s, 1H), 8.8 (s, 1H).

EXAMPLE 4

(4S,2R,3R,5R)-2-{6-amino-2-[4-(4-methylphenyl)pyrazolyl]purin-9-yl}-5-(hydroxymethyl)oxolane-3,4-diol(15)

To a suspension of 2-hydrazinoadenosine (0.025 g, 0.08 mmol) in a 1:1mixture of MeOH/AcOH was added 2-(4-methyl)phenylmalondialdehyde (0.019g, 0.12 mmol) and the mixture was heated at 80° C. for 3 h. Theprecipitate formed was collected by filtration and washed with EtOH andEther to afford 15. ¹HNMR (DMSO-d6) δ3.55 (m, 1H), 3.65 (m, 1H), 3.75(s, 3H), 3.9 (d, 1H), 4.15 (d, 1H), 4.6 (m, 1H), 5.9 (d, 1H), 6.75 (d,2H), 7.6 (d, 2H), 8.15 (s, 1H), 8.35 (s, 1H), 8.8 (s, 1H).

EXAMPLE 5

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-methylcarboxamide(16)

Compound 12 (0.05 mg, 0.12 mmol) was added to 4 mL methylamine (40% sol.In water). The mixture was heated at 65° C. for 24 h. Afterconcentration in vacuo, the residue was purified using prep. TLC (10%MeOH:DCM). ¹HNMR (CD₃OD) 62.90 (s, 3H), 3.78 (m, 1H), 3.91 (m, 1H), 4.13(d, 1H), 4.34 (d, 1H), 4.64 (m, 1H), 6.06 (d, 1H), 8.11 (s, 1H), 8.38(s, 1H), 9.05 (s, 1H).

EXAMPLE 6

1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxylicacid (17)

Compound 12 (0.05 mg, 0.12 mmol) was dissolved in one equivalent of 1NNaOH. The solution was allowed to stir at Rt for 2 h, then acidified topH 4. The resulting precipitate was filtered and washed with water andether. ¹HNMR (CD₃OD) Δ3.75 (m, 1H), 3.90 (m, 1H), 4.13 (d, 1H), 4.43 (d,1H), 4.64 (m, 1H), 6.05 (d, 1H), 8.10 (s, 1H), 8.35 (s, 1H), 9.05 (s,1H).

EXAMPLE 7

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2yl}pyrazol-4-yl)-N,N-dimethylcarboxamide(18)

Compound 18 was prepared in a manner similar to that of compound 16using dimethylamine instead of methylamine, MS 405.12 (M+1).

EXAMPLE 8

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-ethylcarboxamide(19)

Compound 19 was prepared in a manner similar to that of compound 16using ethylamine instead of methylamine, MS 405.35 (M+1).

EXAMPLE 9

1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazole-4-carboxamide(20)

Compound 20 was prepared in a manner similar to that of compound 16using ammonia instead of methylamine, MS 377.25 (M+1).

EXAMPLE 10

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-(cyclopentyl)carboxamide(21)

Compound 12 (0.5 g, 1.2 mmol) was dissolved in dry DMF, TBDMSCl (1.5 g,10 mmol) and imidazole (0.68 g, 10 mmol) were added and the mixture washeated at 80° C. for 24 h. The solvent was evaporated and the residuewas purified by flash column to obtain the trisilyl protected form ofcompound 12. The trisilyl derivative (0.8 g) was then suspended in 1 mLof water and treated with 2 mL1N KOH/MeOH. The mixture was stirred at RTfor 72. The solvent was removed under reduced pressure and the residuewas suspended in 5 mL of water and acidified to pH 5.5 with 1N HCl. Theresulting precipitate was filtered and washed with water and ethyl etherto afford the trisilyl form of the acid 20.

The trisilyl derivative acid 20 (0.14 g, 0.2 mmol) was then dissolved in5 mL dichloromethane. To the solution was added HBTU (0.19 g, 0.4 mmol),HOBt (.076 g, 4 mmol), N-methylmorpholine (0.04 g, 0.4 mmol) and cat.DMAP. The mixture was allowed to stir at RT for 24 h. The mixture wasthen washed with 10% citric acid, saturated NaHCO₃, brine and dried overMgSO₄. The solvent was removed and the residue was treated with 5 mL0.5N NH₄F/MeOH. The solution was heated at reflux for 24 h. The solventwas evaporated and the residue was purified by preparative TLC to affordcompound 21, MS 445.26 (M+1).

EXAMPLE 11

(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)-N-[(4-chlorophenyl)methyl]carboxamide(22)

Compound 22 was prepared in a manner similar to that of compound 21using 4-chlorobenzylamine instead of cyclopentylamine, MS 501.19 (M+1).

EXAMPLE 12

Ethyl 2-[(1-{9-[(4S,2R,3R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-aminopurin-2-yl}pyrazol-4-yl)carbonylamino]acetate(23)

Compound 23 was prepared in a manner similar to that of compound 21using glycine methyl ester instead of cyclopentylamine, MS 445.26 (M+1).

EXAMPLE 13

Compounds of this invention were assayed to determine their affinity forthe A_(2A) receptor in a pig striatum membrane prep. Briefly, 0.2 mg ofpig striatal membranes were treated with adenosine deaminase (2 U/mL)and 50 mM Tris buffer (pH=7.4) followed by mixing. To the pig membraneswas added 2 μL of serially diluted DMSO stock solution of the compoundsof this invention at concentrations ranging from 10 nM to 100 microM orthe control received 2 microL of DMSO alone, then the antagonist ZM241385 in Tris buffer (50 mM, pH of 7.4) was added to achieve a finalconcentration of 2 nM. After incubation at 23° C. for 2 h, then thesolutions were filtered using a membrane harvester using multiplewashing of the membranes (3×). The filter disks were counted inscintillation cocktail to determine the amount of displacement oftritiated ZM displaced by the compounds of this invention. Greater thana 5 point curve was used to generate Ki's. and the number of experimentsis indicated in the column marked in Table 1 below.

TABLE 1 Compound Number A_(2a) Ki, nM n 12 +++ 2 13 ++ 3 14 ++ 1 15 ++ 316 ++ 2 17 − 1 18 +++ 3 19 +++ 3 20 +++ 3 21 +++ 3 22 +++ 3 +++ =10–1,000 nM ++ = 1,000–10,000 nM + = greater than 10,000 nM − = greaterthan 100,000 nM

EXAMPLE 14

The objective of this experiment was to determine the affinities andreceptor binding selectivity of a compound of this invention for A₁,A_(2A), A_(2B) and A₃ adenosine receptors. Molecular cloning hasidentified and confirmed the existence of four subtypes of adenosinereceptors (AdoRs), designated as A₁, A_(2A), A_(2B) and A₃AdoRs (Linden,1994). These AdoR subtypes have distinct anatomical distributions,pharmacological properties and physiological functions (Shryock andBelardinelli, 1997). A₁ and A₃AdoRs couple to inhibitory G proteins(G_(i)/_(o)) and decrease the activity of adenylyl cyclase, whereasA_(2A) and A_(2B)AdoRs increase intracellular cAMP content via couplingto stimulatory G proteins (Gs).

Ligands with high potency and tissue/organ selectivity for distinctadenosine receptor subtypes have therapeutic and diagnostic potentialsfor a variety of diseases (such as arrhythmia, ischemic heart diseases,asthma and Parkinson's disease) and are the focus of considerableresearch efforts by both academia and industry. Here we report thepharmacological and functional characterization of a series of noveladenosine analogues of this invention using mammalian cell linesexpressing either endogenous AdoRs or recombinant human AdoRs.

Materials

Adenosine deaminase was purchased from Boehringer Mannheim BiochemicalsIndianapolis, Ind., U.S.A). [³H]ZM241385 (Lot No. 1) was purchased fromTocris Cookson Ltd (Langford, Bristol, UK). [³H]CPX (Lot No. 3329207)was from New England Nuclear (Boston, Mass., USA). CGS21680 (Lot No.SW-3R-84 and 89H4607), NECA (Lot No. OXV-295E), R-PIA (Lot No. WY-V-23),Rolipram and HEK-hA_(2A)AR membranes were obtained from Sigma-RBI(Natick, Mass.). WRC-0470 was prepared as described in the literature(K. Niiya et al., J. Med. Chem. 35; 4557–4561 (1992); Compound 16 ofthis invention was synthesized as described above and prepared as astock solution (10 mmol/L) in DMSO.

Cell culture and membrane preparation-PC12 cells were obtained from theAmerican Type Culture Collection and grown in DMEM with 5% fetal bovineserum, 10% horse serum, 0.5 mmol/L L-glutamine, 100 U/mL penicillin, 0.1mg/mL streptomycin, and 2.5 μg/mL amphotericin. HEK-293 cells stablyexpressing recombinant human A_(2B)AdoRs (HEK-hA_(2B)AdoR) were grown inDMEM supplemented with 10% fetal bovine serum and 0.5 mg/mL G-418. CHOK1cells stably expressing the recombinant human A₁AdoR (CHO-hA₁AdoR) andA₃AdoR (CHO-hA₃AdoR) were grown as monolayers on 150-mm plastic culturedishes in Ham's F-12 media supplemented with 10% fetal bovine serum inthe presence of 0.5 mg/mL G-418. Cells were cultured in an atmosphere of5% CO₂/95% air maintained at 37° C.

To make membranes, cells were detached from the culture plates intoice-cold 50 mmol/L Tris-HCl buffer (pH7.4). The cell suspensions werehomogenized with Polytron at setting 4 for 30 seconds, and spun at48,000 g for 15 minutes. The pellets were washed three times byre-suspension in ice-cold Tris-HCl buffer and centrifugation. The finalpellet was re-suspended in a small volume of Tris-HCl, aliquoted andfrozen at −80° C. until used for receptor binding assays. The proteinconcentration of membrane suspensions was determined using the Bradfordmethod (Bio-Rad) with bovine serum as standards.

Competition Binding Assays—Competition assays were performed todetermine the affinities (K_(i)) of the following unlabeled compounds(competing agents): Compounds WRC-0470; Compound 16 of this invention,NECA, CGS 21680 and R-PIA for A₁AdoRs ([³H]DPCPX binding sites onCHO-hA₁AdoR cell membranes), A_(2A)AdoRs([³H]ZM241385 binding sites onPC12 and HEK-hA_(2A)AR cell membranes),

A_(2B)AdoR ([³H]DPCPX binding sites on HEK-hA_(2B)AdoR cell membranes)and A₃AdoR ([¹²⁵I]ABMECA binding sites on CHO-hA₃AdoR cell membrane).

Membrane suspensions were incubated for 2 hours at room temperature in50 mmol/L Tris-HCl buffer (pH 7.4) containing ADA (1 U/mL), Gpp(NH)p(100 μM), radioligand {either [³H]ZM241385 (−1.5 to 5 nmol/L), [³H]DPCPX(˜2.5 to 3.0 nmol/L for A₁ and 30 nM for A_(2B)) or [125 I]ABMECA (1nM)}and progressively higher concentrations of the competing agents. Atthe end of incubation, bound and free radioligands were separated byfiltration through Whatman GF/C glass fiber filters using a Brandeltissue harvester (Gaithersburg, Md.). Triplicate determinations wereperformed for each concentration of the competing agent.

Study Design (Protocols)

The affinity (K_(i)) of various CVT compounds for the A₁ and A_(2A)adenosine receptor were determined by their potency to compete for[³H]CPX (A₁) or [3 H]ZM241385 (A_(2A)) binding sites on membranesderived from CHO-hA₁AdoR, PC12 or HEK-HA_(2A)AdoR cells. R-PIA andCGS21680, agonists that are selective for A₁ and A_(2A) respectively,and NECA, a non-selective AdoR agonist were used as controls. Tofacilitate comparison and avoid the complication of multiple affinitystates due to receptor coupling to G-proteins, the competition bindingstudies were carried out in the presence of Gpp (NH) p (100 μM) touncouple receptors from G-proteins. The affinity of selected compoundsfor A_(2B) and A₃ receptors were assessed by their potencies to competefor [3H]CPX (A_(2B)) and [125I]ABMECA (A3) binding sites on membranesderived from HEK-hA_(2B)AdoR and CHO-hA₃AdoR cells, respectively.

Results

The affinity (K_(i)) of WRC-0470; and Compound 16 for human A₁, rat andhuman A_(2A)AdoRs, as determined by competition binding studies aresummarized in Table 2, below. All compounds show moderate selectivityfor human A_(2A) versus A₁ receptor. Furthermore, Compound 16, at aconcentration of 10 μM, decreased the specific binding of [³H]CPX(HEK-hA_(2B)AdoR) or [¹²⁵I]IBMECA (CHO-hA₃AdoR) by 20% and 22%,respectively.

TABLE 2 Binding Affinities of Adenosine Receptor Agonists forA_(2A)AdoRs and A₁AdoRs K_(i)/nmol/L (pK_(i) ± SEM) HEK-hA_(2A)AR CellsCHO-hA₁AR Binding Affinity n Binding Affinity n WRC-0470  272 (6.55 ±0.04) 6    7278 (5.16 ± 0.09) 3 [0.83 ± 0.07] [1.13 ± 0.21] Compound 161269 (5.90 ± 0.03) 7 >16460 (4.59 ± 0.35) 3 [0.73 ± 0.04] [0.92 ± 0.04]CGS21680  609 (6.22 ± 0.06) 3  >3540 (5.47 ± 0.20) 3 {0.65 ± 0.07) NECA 360 (6.45 ± 0.06) 3    328 (6.49 ± 0.06) 3 [0.83 ± 0.08] [0.88 ± 0.03]R-PIA 1656 (5.78 ± 0.02) 3    477 (6.35 ± 0.11) 3 [1.05 ± 0.02) [1.03 ±0.08)

The results of this Experiment show that Compound 16 is a low affinityA_(2A) agonist.

EXAMPLE 15

The objective of this Example was to characterize pharmacologically theeffects of Compound 16 of this invention on coronary artery conductance.Specifically, the experiments were designed to determine 1) the potencyCompound 16 and compare the potency to that of adenosine and otherselected A_(2A) AdoR agonists, and 2) which adenosine receptor, the A₁or A_(2A) AdoR subtype mediates the coronary vasodilation caused byCompound 16 of this invention.

In the heart, the A_(2A) adenosine receptor mediates the coronaryvasodilation caused by adenosine, whereas the A₁ receptor mediates thecardiac depressant actions of adenosine, such as the negativechronotropic and dromotropic (AV block) effects.

Several potent and selective ligands, both agonists and antagonists, forthe A₁ and A_(2A) AdoRs have been synthesized. In the heart agonists ofA₁ AdoRs have been proposed to be useful as antiarrhythmic agents,whereas agonists of A_(2A) AdoRs are being developed for selectivecoronary vasodilation

A series of adenosine derivatives targeted for selective activation ofA_(2A) adenosine receptor (A_(2A) AdoR) were synthesized for thepurposes of developing coronary vasodilators. More specifically, in thisstudy we report on the effect of a series of novel A_(2A) AdoR agonistson coronary artery conductance (vasodilation) in rat and guinea pigisolated perfused hearts.

Materials

Rats (Sprague Dawley) and Guinea pigs (Hartley) were purchased fromSimonsen and Charles Rivers, respectively. WRC-0470 was prepared asdescribed in the literature (K. Niiya et al., J. Med. Chem. 35;4557–4561 (1992). Compound 16 of this invention was prepared asdescribed above. CGS 21680 and adenosine were purchased from Sigma.Krebs-Henseleit solution was prepared according to Standard Methods, and0.9% saline was purchased from McGraw, Inc.

Methods

Adult Sprague Dawley rats and Hartley guinea pigs of either sex weighingfrom 230 to 260 grams and 300 to 350 grams, respectively were used inthis study. Animals were anesthetized by peritoneal injection of acocktail containing ketamine and xylazine (ketamine 100 mg, xylazine 20mg/ml). The chest was opened and the heart quickly removed. The heartwas briefly rinse in ice-cold Krebs-Henseleit solution (see below), andthe aorta cannulated. The heart was then perfused at a flow rate of 10ml/min with modified Krebs-Henseleit (K-H) solution containing NaCl117.9, KCl 4.5, CaCl₂ 2.5, MgSO₄ 1.18, KH₂PO₄ 1–18, pyruvate 2.0 mmo/L.The K-H solution (pH 7.4) was gassed continuously with 95% O₂ and 5% CO₂and warmed to 35±0.50° C. The heart was electrically paced at a fixedcycle length of 340 ms (250 beats/min) using a bipolar electrode placeon the left atrium. The electrical stimuli were generated by a Grassstimulator (Model S48, W. Warwick, R.I.) and delivered through a StimuliIsolation Unit (Model SIU5, Astro-Med, Inc., NY) as square-wave pulsesof 3-msec in duration and amplitude of at least twice the thresholdintensity.

Coronary perfusion pressure (CPP) was measured using a pressuretransducer, connected to the aortic cannula via a T-connector positionedapproximately 3 cm above the heart. Coronary perfusion pressure wasmonitored throughout the experiment and recorded either on a chartrecorder (Gould Recorder 2200S) or a computerized recording system(PowerLab/4S, ADinstruments Pty Ltd, Australia). Only hearts with CPPranging from 60 to 85 mmHg (in the absence of drugs) were used in thestudy. Coronary conductance (in ml/min/mmHg) was calculated as the ratiobetween coronary perfusion rate (10 ml/min) and coronary perfusionpressure.

In experiments in which A₁ adenosine receptor-mediated negativedromotropic effect was measured, atrial and ventricular surfaceelectrograms were recorded during constant atrial pacing. The effect ofvarious adenosine receptor agonists on atrioventricular conduction timewas determined as described previously by Jenkins and Belardinelli Circ.Res. 63: 97–116 (1988).

Stock solutions of Compound 16 of this invention (5 mM) and CGS 21680 (5mM) were prepared in dimethyl sulfoxide (DMSO); purchased from Aldrich,PS 04253MS. A stock solution of adenosine (1 mg/ml) was prepared insaline. One concentration was made from the stock solution by dilutioninto saline to yield solution of either 2×10⁻⁴ or 2×10⁻⁵ M. Thesesolutions were injected into the perfusion line of the apparatus asboluses of 20 μl. In some experiments the solutions were placed into a30 ml glass syringe and the drugs were infused at rates necessary toachieve the desired perfusate concentrations (e.g, 10, 100 nM, etc).

Coronary Vasodilation of A_(2A) Adenosine Receptor Agonists

Concentration-response relationships for the effect of Compound 16 ofthis invention (0.1 to 400 nM) and CGS21680 (0.1 to 10 nM) to increasecoronary conductance were obtained. After control measurements ofcoronary perfusion pressure were recorded, progressive higherconcentrations of the adenosine receptor agonists were administereduntil maximal coronary vasodilation was observed. The steady-stateresponses to each concentration of adenosine receptor agonists wererecorded. In each heart of this series (4 to 6 hearts for each agonist)only one agonist and one concentration-response relationship wasobtained.

Coronary Vasodilatory Effect of Compound 16 in the Absence and Presenceof Adenosine Receptor Antagonists.

To determine which adenosine receptor subtype (A₁ or A_(2A)) mediatesthe coronary vasodilation caused by Compound 12, the A₁ and A_(2A)adenosine receptor antagonists CPX and ZM241385, respectively, wereused. Hearts (n=6) were exposed to the compound being tested (10 nM),and after the effect of this agonist reached steady-state, first CPX (60nM), and then ZM241385 were added to the perfusate and the changes inCPP were recorded.

In isolated perfused hearts (n=36 rats and 18 guinea pigs) paced atconstant atrial cycle length of 340 msec, adenosine, CGS21680, WRC0470,and Compound 16 caused a concentration-dependent increase in coronaryconductance. CGS21680 and WRC0470 were the most potent agonists tested.Compound 16 was approximately 10-fold more potent than adenosine toincrease coronary conductance. It is worth noting that all agonists wereseveral fold more potent coronary vasodilators in rat than guinea pighearts (Table 3).

TABLE 3 Potency of Adenosine and A_(2A) Adenosine Receptor Agonists toIncrease Coronary Conductance in Rat and Guinea Pig Isolated PerfusedHearts Potency (EC₅₀) Agonist n Rat Guinea Pig Compound 16 4 6.4 ± 1.218.6 ± 6.0  Adenosine 4 59.2 ± 6.4  86.0 ± 0.5  CGS21680 4 0.5 ± 0.1 1.7± 0.4 WRC0470 3 0.6 ± 0.2 2.4 ± 1.1

To determine the AdoR subtype (A₁ versus A_(2A)) that is responsible forthe coronary vasodilation observed in the presence of Compound 16, theeffect of this agonist (10 nM) on coronary conductance was studied inthe absence and presence of CPX, a selective A₁ AdoR antagonist(Belardinelli et al, 1998) and ZM241385, a selective A_(2A) AdoRantagonist (Poucher et al, 1995) at the concentration of 60 nM. As shownin FIG. 1, Compound 16 significantly increased coronary conductance to0.22+0.01 ml/mm Hg⁻¹ min^(−′) from a baseline value of 0.16+0.02mlmmHg⁻¹min⁻¹. This increase in coronary conductance caused by Compound16 was not affected by CPX but was completely reversed by ZM241385(0.17±0.02 mlmm Hg-1 min⁻′).

EXAMPLE 16

The objective of this Example was to determine the functionalselectivity of Compound 16 to cause coronary vasodilation. Specifically,the potency of Compound 16 to cause coronary vasodilation (A_(2A) AdoRresponse) and prolongation of A-V nodal conduction time (A₁ AdoRresponse) were determined in rat and guinea pig hearts.

Materials

Sprague Dawley rats were purchased from Simonsen. Hartley guinea pigswere purchased from Charles River. Compound 16 was prepared as describedabove.CVT-510-2-{6-[((3R)oxolan-3-yl)amino]purin-9-yl}(4S,3R,5R)-5-(hydroxymethyl)oxolane-3,4-diol—wasprepared in accordance with the synthesis method disclosed in U.S. Pat.No. 5,789,416, the specification of which is incorporated herein byreference. Ketamine was purchased from Fort Dodge Animal Health (Lot No.440444) and xylazine from Bayer (Lot No. 26051 A). Krebs-Henseleitsolution was prepared according to the standard methods, and 0.9% sodiumchloride was purchased from McGraw, Inc. (Lot No. J8B246).

Isolated Perfused Heart Preparation:

Rats and guinea pigs, of either sex weighing from 230 to 260 grams and300 to 350 grams, respectively, were used in this study. Animals wereanesthetized by peritoneal injection of a cocktail containing ketamineand xylazine (ketamine 100 mg, xylazine 20 mg/ml). The chest was openedand the heart quickly removed. The heart was briefly rinse in ice-coldKrebs-Henseleit solution (see below), and the aorta cannulated. Theheart was then perfused at a flow rate of 10 ml/min with modifiedKrebs-Henseleit (K-H) solution containing NaCl 117.9, KCl 4.5, CaCl₂2.5, MgSO₄ 1.18, KH₂PO₄ 1.18, pyruvate 2.0 mmo/L. The K-H solution (pH7.4) was gassed continuously with 95% O₂ and 5% CO₂ and warmed to35±0.5.° C. The heart was electrically paced at a fixed cycle length of340 ms (250 beats/min) using a bipolar electrode place on the leftatrium. The electrical stimuli were generated by a Grass stimulator(Model S48, W. Warwick, R.I.) and delivered through a Stimuli IsolationUnit (Model SIU5, Astro-Med, Inc., NY) as square-wave pulses of 3-msecin duration and amplitude of at least twice the threshold intensity.

Coronary perfusion pressure (CPP) was measured using a pressuretransducer, connected to the aortic cannula via a T-connector positionedapproximately 3 cm above the heart. Coronary perfusion pressure wasmonitored throughout the experiment and recorded either on a chartrecorder (Gould Recorder 2200S) or a computerized recording system(PowerLab/4S, ADInstruments Pty Ltd, Australia). Only hearts with CPPranging from 60 to 85 mmHg (in the absence of drugs) were used in thestudy. Coronary conductance (in m1/min/mmHg) was calculated as the ratiobetween coronary perfusion rate (10 ml/min) and coronary perfusionpressure.

A₁ adenosine receptor-mediated depression of A-V nodal conduction time(negative dromotropic effect) was measured. Atrial and ventricularsurface electrograms in rats and His bundle electrogram in guinea pigs,were recorded during constant atrial pacing. The effects of Compound 16on atrioventricular conduction time and stimulus-to-His-bundle (S-Hinterval) were determined as described previously by Jenkins andBelardinelli (1988).

The effects of Compound 16 on coronary conductance (A_(2A) effect) andatrioventricular conduction time or stimulus-to-His-bundle (S-H)interval (A₁ effect) was then determined. Hearts were instrumented forcontinuous recording of coronary perfusion pressure (A_(2A) response)and atrioventricular (A-V) conduction time or S-H interval (A₁response). In each experiment, concentration-response relationship ofCompound 16 (n=6 rats, 4 guinea pigs) to increase coronary conductanceand to prolong A-V conduction time or S-H interval was determined. Aftercontrol measurements of CPP and A-V conduction time or S-H interval weremade, progressive higher concentrations of Compound 16 was administereduntil maximal coronary vasodilation and A-V nodal conduction time or S-Hinterval prolongation were achieved. In separate rat hearts (n=4), theeffect of various concentrations (100–400 nM) of CVT510, an A₁ adenosineagonist (Snowdy et al, 1999), on A-V nodal conduction time wasdetermined and compared to that of Compound 16 (0.1–30 μM).

The concentration-response curves for Compound 16 to increase coronaryartery conductance and to prolong A-V nodal conduction time or S-Hinternal are shown in FIGS. 2 and 3. In both rat and guinea pig,Compound 16 increased coronary conductance in a concentration dependentmanner. The potencies (EC₅₀ values) for Compound 16 to increase coronaryconductance in rat hearts was 6.4±0.6 nM and in guinea pig hearts was18.6±6.0 nM. In contrast, the effect of this agonist on S-H interval wassomewhat variable between rat and guinea pig hearts. In rat heartsCompound 16 did not prolong A-V nodal conduction time (FIGS. 2 and 3)whereas the A₁ AdoR agonist CVT510 significantly prolonged the A-V nodalconduction time (FIG. 4). Unlike in rat, in guinea pig hearts Compound16 caused a concentration-dependent prolongation of S-H interval (A1response) with an EC₅₀ value (potency) of 4.0±2.3 μM (FIG. 4). Thislatter value is approximately 215-fold greater (i.e., less potent) thanthe EC₅₀ value of 18.6±6.0 nM to cause coronary vasodilation (A_(2A)response—FIG. 3).

The results indicate that Compound 16 is a coronary vasodilator (A_(2A)AdoR-mediated effect) devoid of negative dromotropic effect (A₁AdoR-mediated effect) in rat hearts. In guinea pig hearts Compound 16caused some negative dromotropic effect. Nevertheless, Compound 16 wasat least 215-fold more selective to cause coronary vasodilation thannegative dromotropic effect. The reason(s) for the species difference inthe A₁ AdoR-mediated response elicited by Compound 16 is unknown.Regardless, in both species (rat and guinea pig) Compound 16 causesmaximal coronary vasodilation at concentrations that do not causeprolongation of A-V nodal conduction time, i.e., without negativedromotropic effect. It was also observed that Compound 16 has a greateraffinity (i.e., >2-/>-13-fold) for A_(2A) than A₁ AdoR and that there isa markedly greater receptor reserve for A_(2A) AdoR-mediated coronaryvasodilation than for A₁ AdoR-mediated negative dromotropic effect.

EXAMPLE 17

The present study was designed to test the hypothesis that there is aninverse relationship between the affinity (K_(i) or pK_(i)) and durationof action of A_(2A) adenosine receptors (AdoR). Specifically, the aimsof the study were to determine the relationship between the duration ofthe coronary vasodilation caused by a selected series of high and lowaffinity A_(2A)AdoR agonists in rat isolated hearts and anesthetizedpigs; and the affinity of these agonists for A_(2A) AdoRs in pigstriatum.

Materials: Rats (Sprague Dawley) were purchased from Simonen. Farm pigswere obtained from Division of Laboratory Animal Resources, Universityof Kentucky. Compound 12, Compound 13, and Compound 16 of this inventionwere prepared as described in the methods above. YT-0146 was prepared asdescribed in U.S. Pat. No. 4,956,345, the specification of which isincorporated herein by reference. WRC-0470 was prepared as described inthe literature (K. Niiya et al., J. Med. Chem. 35; 4557–4561 (1992).CGS21680 was purchased from Research Biochemicals, Inc. and Sigma andR-PIA (Lot No. WY-V-23) was purchased from Research Biochemicals, Inc.HENECA was a gift from Professor Gloria Cristalli of University ofCamerino, Italy.

The anesthetic agents: Ketamine was purchased from Fort Dodge AnimalHealth. Xylazine was purchased from Bayer. Sodium pentobarbital waspurchased from The Butler Co. Phenylephrine was purchased from Sigma.DMSO was purchased from Sigma and American Tissue Type Collections.Krebs-Henseleit solution was prepared according to standard methods, and0.9% saline was purchased from McGraw, Inc.

In this study, the following laboratory preparations were used. 1) Ratisolated perfused hearts; 2) Anesthetized open-chest pigs;

Rat Isolated Perfused Heart Preparation

Adult Sprague Dawley rats of either sex weighing from 230 to 260 gramswere used in this study. Animals were anesthetized by peritonealinjection of a cocktail containing ketamine and xylazine (ketamine 100mg, xylazine 20 mg/ml). The chest was opened and the heart quicklyremoved. The heart was briefly rinse in ice-cold Krebs-Henseleitsolution (see below), and the aorta cannulated. The heart was thenperfused at a flow rate of 10 ml/min with modified Krebs-Henseleit (K-H)solution containing NaCl 117.9, KCl 4.5, CaCl. 2.5, MgSO₄ 1.18, KH₂PO₄1.18, pyruvate 2.0 mmo/L. The K-H solution (pH 7.4) was gassedcontinuously with 95% O₂ and 5% CO₂ and warmed to 35±0.50° C. The heartwas electrically paced at a fixed cycle length of 340 ms (250 beats/min)using a bipolar electrode place on the left atrium. The electricalstimuli were generated by a Grass stimulator (Model S48, W. Warwick,R.I.) and delivered through a Stimuli Isolation Unit (Model SIU5,Astro-Med, Inc., NY) as square-wave pulses of 3msec in duration andamplitude of at least twice the threshold intensity.

Coronary perfusion pressure (CPP) was measured using a pressuretransducer, connected to the aortic cannula via a T-connector positionedapproximately 3 cm above the heart. Coronary perfusion pressure wasmonitored throughout the experiment and recorded either on a chartrecorder (Gould Recorder 2200S) or a computerized recording system(PowerLab/4S, ADInstruments Pty Ltd, Australia). Only hearts with CPPranging from 60 to 85 mmHg (in the absence of drugs) were used in thestudy. Coronary conductance (in ml/min/mmHg) was calculated as the ratiobetween coronary perfusion rate (10 ml/min) and coronary perfusionpressure.

Anesthetized Open-chest Pig Preparation

Farm pigs weighing 22–27 kg were used in this study. All animalsreceived humane care according to the guidelines set forth in “ThePrinciples of Laboratory Animal Care” formulated by the National Societyfor Medical research and the “Guide for the Care and Use of LaboratoryAnimals” prepared by the Institute of Laboratory Animal Resources andpublished by the National Institutes of Health (NIH Publication No.86-23, revised 1996). In addition, animals were used in accordance withthe guidelines of the University of Kentucky Institutional Animal Careand Use Protocol.

Anesthesia was anesthetized with ketamine (20 mg/kg, i.m.) and sodiumpentobarbital (15–18 mg/kg i.v.). Anesthesia was maintained withadditional sodium pentobarbital (1.5–2 mg/kg, i.v.) every 15–20 minutes.Ventilation was maintained via a tracheotomy using a mixture of room airand 100% O₂. Tidal volume, respiratory rate and fraction of O₂ ininspired air were adjusted to maintain normal arterial blood gas (ABG)and pH values. Core body temperature was monitored with an esophagealtemperature probe and maintained with a heating pad between 37.0–37.5°C. Lactate Ringers solution was administered via an ear or femoral vein,at 5–7 ml/kg/min after a initial bolus of 300–400 ml. A catheter wasinserted into the femoral artery to monitor arterial blood pressure andto obtain ABG samples.

The heart was exposed through a median stemotomy, and suspended in apericardial cradle. Left ventricular pressure (LVP) was measured with a5F high fidelity pressure sensitive tip transducer (Millar Instruments,Houston, Tex.) placed in the left ventricular cavity via the apex andsecured with a purse string suture. A segment of the left anteriordescending coronary artery (LAD), proximal to the origin of the firstdiagonal branch, was dissected free of, surrounding tissue. A transittime perivascular flow probe (Transonic Systems Inc., Ithaca, N.Y.) wasplaced around this segment to measure coronary blood flow (CBF).Proximal to the flow probe a 24 g modified angiocatheter was insertedfor intracoronary infusions. All hemodynic data were continuouslydisplayed on a computer monitor and fed through a 32 bit analog-digitalconverter into an online data acquisition computer with customizedsoftware (Augury, Coyote Bay Instruments, Manchester, N.H.). A_(2A) AdoRagonists were dissolved in DMSO to produce stock concentrations of 1–5mM, which were diluted in 0.9% saline and infused at rates of 1–1.5ml/min. The A_(2A) AdoR agonists were administered intracoronary. Tomaintain blood pressure constant, phenylephrine was administeredintravenously. The phenylephrine stock solution (30 mM) was prepared indistilled water.

Isolated Perfused Hearts

To determine the duration of the A_(2A)adenosine receptor mediatedcoronary vasodilation caused by adenosine and adenosine receptoragonists, the agonists were administered interveneously either by bolusinjection (protocol A) or by continuous infusion (protocol B).

Protocol A: Bolus injections. In each heart of this series (3 to 11hearts for each agonist), boluses of adenosine (20 μl, 2×10⁻⁴ M),Compounds of this invention (20 to 40 μl, 2×10⁻⁵ M), and other adenosinereceptor agonists were injected into the perfusion line. The times to50% (t 0.5) and 90% (t 0.9) reversal of the decrease in CPP weremeasured. Each heart was exposed to a maximum of three vasodilators.

Protocol B: Continuous infusion. In a separate series of experiments(n=4), Compound 16 and adenosine were infused into the perfusion line atconstant rate for a period of six minutes. The perfusate concentrationsof Compound 16 and adenosine were 20 nM and 200 nM respectively, whichwere approximately 4× their respective concentrations previouslyestablished to cause 50% of maximal increase in coronary conductance(EC₅₀) in rat isolated perfused hearts. The times to 50% (t 0.5) and 90%(t 0.9) reversal of the decreases in CPP were measured from the time atwhich the infusion of the agonists was stopped.

Dose-dependent duration of maximal vasodilation caused by bolusinjections of Compound 16. To determine the dependency of the durationof maximal coronary vasodilation on the dose of Compound 16, boluses(100–300 μl) of a 2×10⁻⁵ M stock solution of Compound 16 were injectedinto the perfusion line. In addition, the duration of the injection wasvaried according to the volume of the boluses such as 10, 20 and 30 secfor 100, 200 and 300 μl boluses respectively. The duration of maximaleffect was measured from the point at which the decrease in CPP reachedthe nadir to the onset point of reversal of CPP.

Relationship between affinity of various agonists for A_(2A) adenosinereceptor and the reversal time of their effect to increase coronaryconductance: These experiments were performed to construct therelationship between the affinities of the various agonists forA_(2A)adenosine receptor and the duration of their respective effect oncoronary conductance. Boluses of various agonists were injected into theperfusion line of rat isolated perfused hearts (n=4 to 6 for eachagonist) and the time to 90% (t 0.9) reversal of the decrease in CPPmeasured. The affinities of the various agonists for A_(2A) adenosinereceptor was determined in pig striatum membranes using a radioligandbinding assay, as described above. The reversal time (t 0.9) of thedecrease in CPP was plotted against their affinities (pK_(i)) for theA_(2A) adenosine receptor.

Open-chest Pig

Prior to initiating the experiment, a 30-minute stabilization periodfollowed the completion of all instrumentation. After obtaining thebaseline hemodynamic data the first intracoronary infusion of an A_(2A)ADOR agonist was initiated. Infusions were maintained for 4–5 minutes toallow LAD CBF to reach a steadystate, after which the infusion wasterminated. The time to recovery of 50% (t 0.5) and 90% (t 0.9) ofbaseline CBF were recorded. Ten to 15 minutes after CBF returned topre-drug values a second infusion with a different agonist was started.In preliminary studies it was found that the intracoronary infusion ofadenosine agonists produced varying degrees of systemic hypotension, andhence, in all subsequent experiments, phenylephrine was administeredintravenously. Hemodynamic measurements were made prior to and followingthe initiation of the phenylephrine infusion at dose of—1 μg/kg/min. Thephenylephrine infusion rate was adjusted during and following theinfusions of the adenosine agonists to maintain arterial blood pressurewithin 5 mmHg of preinfusion values. The effect of a maximum of threedifferent agonists was determined in each experiment.

Results

Adenosine, the compounds of this invention and other adenosinederivatives were given as boluses into the perfusion line atconcentrations that cause equal or near-equal increases in coronaryconductance. Although adenosine and the agonists caused equal maximalincreases in coronary conductance the duration of their effect wasmarkedly different. The duration of the effect of adenosine was theshortest followed by Compound 16, whereas that of CGS21680 and WRC0470were the longest. The durations of the coronary vasodilation caused byadenosine, the compounds of this invention and other agonists measuredas the time to 50% and 90% (t 0.5 and t 0.9, respectively) reversal ofthe increases in coronary conductance are summarized in Table 4

TABLE 4 Reversal Time Of Coronary Vasodilation by Adenosine andadenosine receptor agonists in Rat Isolated Perfused Hearts Agonist t0.5 (min) t 0.9 (min) n Adenosine  1.06 ± 0.1  5.6 ± 0.8 11 HENECA  28.6± 1.1 32.8 ± 3.1 3 R-PIA  7.9 ± 0.1 12.6 ± 0.8 3 CGS21680  14.5 ± 0.919.5 ± 0.9 3 YT-146  17.7 ± 1.0 28.5 ± 4.0 3 Compound 12 14.83 ± 2.115.0 ± 0.8 3 Compound 13  14.4 ± 1.9 21.3 ± 3.9 4 Compound 16  5.2 ± 0.211.3 ± 1.1 5 Time (in minutes) to 50% and 90% (t 0.5 and t 0.9,respectively) reversal of the increases in coronary conductance causedby adenosine and adenosine receptor agonists. Values are the means ± SEMof single determinations in each of the preparations (n).The reversal time of coronary vasodilation was dependent on the affinityof the adenosine derivatives for brain striatum A_(2A) receptors. (FIG.2A) There was a significant (P<0.05) inverse relationship (r=0.87)between the affinity (PKi) of the agonists for the A_(2A)AdoR and thereversal time (t 0.9) of the coronary vasodilation caused by the sameagonists.

Regardless of whether Compound 16 was given as bolus or continuousinfusion the reversal of the coronary vasodilation was relatively rapid.In fact, a comparison between a six minute infusion of adenosine andCompound 16 at doses that they cause equal decreases in coronaryperfusion pressure (CPP) revealed that adenosine and Compound 16 have asimilar time course for vasodilation and reversal time. Both the t 0.5and t 0.9 were near identical. The duration of the coronary vasodilationby Compound 16 was dose-dependent. Increasing the volume of a bolus ofCompound 16 (stock solution of 2×10⁻⁵ M) caused progressively longerlasting decreases in CPP. The maximal duration of the coronaryvasodilation (time that CPP remained at its lowest) increased as thevolume of the boluses increased from 100 μl to 200 and 300 μl withoutaffecting the maximal decreases in CPP.

Coronary Vasodilation in an Open-chest Pig Preparation

In in situ hearts of an open-chest anesthetized pig preparation Compound16 of this invention as well as CGS21680 and other A_(2A)AdoR agonists(i.e., WRC-0470 and YT-146) caused significant increases in coronaryblood flow (CBF). Selected doses of these compounds given as continuous(4 to 5 min) intracoronary infusions caused 3.1 to 3.8-fold increases inCBF as set forth in Table 3, below. Once established that all agonistscaused near the same magnitude of increases in CBF (i.e., “foldincrease”) and cause similar changes in heart rate and mean arterialblood pressure, the reversal time of their respective coronaryvasodilation effects was determined.

TABLE 5 Magnitude of Increase in Coronary Blood Flow Caused by VariousAdenosine Receptor Agonists in Open-Chest Anesthetized Pigs Agonist CBF(“Fold Increase” n Compound 16 (10 μg/kg/min) 3.40 ± 0.04 3 Compound 16(310 μg/kg/min) 3.83 ± 0.39 6 WRC-470 (1 μg/kg/min) 3.14 ± 0.24 6GSC21680 (2 μg/kg/min)  3.54 ± 0.093 3 YT-146 (1 μg/kg/min) 3.44 ± 0.473 Maximal “fold-increase” in coronary blood flow (CBF) above baselinecaused by various adenosine receptor agonists. Data represent mean ± SEMof one or two measurements in each pig (n).As summarized in Table 6 the t_(0.5) and t_(0.9) of coronaryvasodilation caused by the various A_(2A) AdoR agonists and“CVT-compounds” was variable. The reversal time of the increase in CBFcaused by Compound 16 of this invention were shorter than that ofCGS21680, WRC-0470 or YT-146. More importantly, as in rat isolatedperfused hearts, there was a significant (P<0.05) inverse relationship(r=0.93) between the affinity (PKi) of the A_(2A)AdoR agonists for pigbrain striatum A_(2A) receptors and the reversal time (t 0.9) ofcoronary vasodilation. There was an excellent concordance between thereversal time of the coronary vasodilation caused by a selected numberof agonists in rat isolated perfused hearts and in anesthetized openchest pig preparations.

TABLE 6 Reversal Time of Coronary Vasodilation Caused by VariousAdenosine Receptor Agonists in Open-Chest Anesthetized Pigs Agonistt_(0.5) (min) t_(0.9) (min) n Compound 16 (10 μg/kg/min) 1.9 ± 0.2 10.1± 0.7 3 Compound 16 (310 μg/kg/min) 2.6 ± 0.4 12.3 ± 1.1 6 WRC-470 (1μg/kg/min) 9.5 ± 0.8 22.5 ± 1.6 6 GSC21680 (2 μg/kg/min) 9.7 ± 0.8 21.4± 0.8 3 YT-146 (1 μg/kg/min) 17.8 ± 3.4  32.9 ± 5.6 3 Time (in minutes)to 50% and 90% (t_(0.5) and t_(0.9), respectively) reversal of theincreases in coronary blood flow caused by adenosine receptor agonists.Values are the means ± SEM of one or two determinations in each animal(n).

Compound 16 is a low affinity A_(2A)AdoR agonists and less potent(−10-fold) than the prototypical agonist CGS21680. Nevertheless Compound16 is a full agonist to cause coronary vasodilation. But, as shown inthis study the duration of its effect is several-fold shorter than thatof the high affinity agonists CGS21680 and WRC-0470. Hence, Compound 16is a short acting A_(2A) AdoR agonists coronary vasodilator. Because ofits short duration of action in comparison to the high affinityA_(2A)AdoR agonists (e.g., WRC-0470, CGS21680) this low affinity butstill full agonist coronary vasodilator may prove to be idealpharmacological “stressor agents” during radionuclide imaging of themyocardium.

Table 7 lists the A_(2A) adenosine agonists and antagonists that wereused in Examples 14–18.

TABLE 7 Biologically Active A_(2a) Adenosine Receptor Agonists andAntagonists. Compound Abbreviation Activity5′-N-ethylcarboxamidoadenosine NECA A_(2A) agonistN-[(1R)-1-methyl-1\2-phenylethyl]adenosine R-PIA A_(2A) agonist8-cyclopentyl-1,3-dimethylxanthinge CPX A_(2b) antagonist4-[2-[[6-Amino-9-(ethyl-B-D ribofuranuronamindosyl)- CGS21680 A_(2A)9H-purin-2-yl]aminoethyl]benzenepropanoic adenosine acid receptoragonist N-ethyl-1′-deoxy-1′-(6-amino-2-hexynyl-9H- HENECA A_(2a)adenosine purin9-yl)-beta-D-ribofura nuronamide receptor agonist2-alkynyladenosine YT-0146 A_(2a) adenosine receptor agonist2-cyclohexylmethylidenehydrazinoadenosine WRC0470 receptor agonist4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3- ZM241385 A_(2A) adenosinea] receptor [1,3,5]triazin-5-ylamino]ethyl)phenol antagonist

1. A compound having the formula:

wherein R₁=CH₂OH; R₂ and R₄ are each hydrogen; R₃ is —CONR⁷R⁸ R₇ isselected from the group consisting of hydrogen and C₁₋₃ alkyl; and R₈ isselected from the group consisting of hydrogen and C₁₋₈ alkyl.
 2. Thecompound of claim 1 wherein R₇ is hydrogen and R₈ is C₁₋₈ alkyl.
 3. Thecompound of claim 1 wherein R₇ is C₁₋₃ alkyl and R₈ is hydrogen.
 4. Thecompound of claim 1 wherein R₇ is C₁₋₃ alkyl and R₈ is C₁₋₈ alkyl. 5.The compound of claim 1 wherein R₇ is hydrogen and R₈ is hydrogen. 6.The compound of claim 1 wherein R₇ is selected from the group consistingof hydrogen and C₁₋₂ alkyl.
 7. The compound of claim 1 wherein R₇ ishydrogen.
 8. The compound of claim 1 wherein R₈ is hydrogen.
 9. Thecompound of claim 1 wherein R₈ is methyl.
 10. A method for stimulatingcoronary vasodilation in a mammal by administering to the mammal in needthereof a therapeutically effective amount of the compound of claim 1.11. The method of claim 10 wherein the mammal is a human.
 12. The methodof claim 10, wherein the therapeutically effective amount of thecompound is sufficient to stress the heart and induce a coronary stealsituation.
 13. The method of claim 10, wherein the therapeuticallyeffective amount of the compound is sufficient for imaging heart orcoronary activity in the mammal.
 14. The method of claim 10 wherein thecompound is administered by iv bolus.
 15. The method of claim 14 whereinthe compound is administered in from 10 to 30 seconds.
 16. A method ofproducing coronary vasodilation without peripheral vasodilationcomprising administering a compound of claim 1 to a human in needthereof.
 17. The method of claim 16 wherein the compound is administeredby iv bolus.
 18. The method of claim 16 wherein the compound isadministered in from 10 to about seconds.
 19. The method of claim 16,wherein the compound is administered in an amount that is sufficient forimaging heart or coronary activity.
 20. A method of myocardial perfusionimaging of a human, comprising administering a radionuclide and acompound of claim 1 to the human in need thereof wherein the myocardiumis examined for areas of insufficient blood flow followingadministration of the radionuclide and the compound.
 21. The method ofclaim 20, wherein the amount of the compound is sufficient to stress theheart and induce a coronary steal situation.
 22. The method of claim 20wherein the compound is administered by iv bolus.
 23. The method ofclaim 22 wherein the compound is administered in from 10 to 30 seconds.24. The method of claim 20 wherein the compound is administered beforethe radionucleotide is administered.
 25. A pharmaceutical compositioncomprising a compound of claim 1 and at least one pharmaceuticalexcipient.
 26. The pharmaceutical composition of claim 25 wherein thepharmaceutical composition is in the form of a solution.
 27. Thepharmaceutical composition of claim 25 wherein the pharmaceuticalcomposition is formulated for injection.